Fig. 8

Western immunoblotting of CDA, DNMT1, p53, JAK1, Caspase-3&7, PARP and PPARγ in colon cancer cells following CDA gene knockdown and its pharmacological inhibition by THU. A1 Western blotting of CDA and DNMT1 following decitabine treatment of colon cancer cells. Decitabine treatment of Caco-2, HCT-116-p53wt and HCT-116-p53null cells repressed DNMT1 protein expression; however, it caused a small but significant induction of the CDA protein. Gene knockdown of CDA in decitabine treated colon cancer cells repressed CDA and DNMT1 protein expression. The quantification of the WB experiments is shown as histogram. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. A2 Regulation of CDA gene expression following decitabine, PBA and the combined drug treatment of Caco-2 cells for 96 h. The CDA transcript expression remained unchanged following drug treatment of Caco-2 cells. A3 CDA gene knockdown in Caco-2, HCT-116-p53wt, and HCT-116-p53null cells. Shown are RT-qPCR assays after treatment of colon cancer cell lines with single-stranded antisense oligonucleotides (ASO) for 72 h. The GapmeRs ASO treatment of colon cancer cells repressed CDA gene expression significantly. Statistical significance: **p < 0.01, ***p < 0.001. B Western blotting of CDA, DNMT1, p53 and JAK1 protein after decitabine and THU treatment of Caco-2, HCT-116-p53wt and HCT-116-p53null cells for 72 h. We used 20, 40 and 80 μg/ml THU drug concentrations to inhibit CDA activity in decitabine treated colon cancer cell lines. Decitabine at 3 µM concentrations caused a small but significant increase in CDA protein. Importantly, drug treatment induced p53 and repressed DNMT1 and JAK1 protein expression. The combined decitabine/THU treatment at various drug concentrations had no effect on CDA; nonetheless, significantly induced p53 and repressed JAK1 protein expression. The quantification of the WB experiments is shown as histogram. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C Immunoblotting of Caspase-3, -7, PARP and PPARγ protein in response to decitabine, PBA and the combined drug treatment of colon cancer cells for 72 h. Decitabine treatment of Caco-2, HCT-116-p53wt and HCT-116-p53null cells repressed Caspase-3, -7 and PARP protein; however, induced expression of the cleaved products and PPARγ protein. Thus, decitabine treatment induced programmed cell death. Likewise, PBA treatment of colon cancer cells significantly repressed Caspase-3, -7 and PARP protein expression; however, it induced the cleaved products and PPARγ protein (C1). Importantly, in CDA silenced colon cancer cell lines, the effects of the combined decitabine/PBA treatment on apoptosis were enhanced. The quantification of the WB experiments is shown as histogram (C2). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. D Immunoblotting of Caspase-3&7, PARP and PPARγ following the combined decitabine/PBA and THU treatment of colon cancer cells. The combined decitabine/PBA treatment of Caco-2, HCT-116-p53wt and HCT-116-p53null cell lines repressed expression of caspase-3&7 and PARP protein; however, it induced expression of the cleaved products and PPARγ. Thus, decitabine/PBA treatment induced programmed cell death (D1). Pharmacological inhibition of CDA at two different THU drug concentrations significantly enhanced the effects of combined decitabine/PBA treatment. The quantification of the WB experiments is shown as histogram (D2). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001